Overexpression of manganese superoxide dismutase suppresses tumor formation by modulation of activator protein-1 signaling in a multistage skin carcinogenesis model.

Manganese superoxide dismutase (MnSOD) is a nuclear encoded primary antioxidant enzyme localized in mitochondria. Because expression of MnSOD plays a major role in maintaining cellular redox status and reactive oxygen species are known to play a role in signal transduction and carcinogenesis, we investigated the role of MnSOD in the development of cancer using a two-stage [7,12-dimethylbenz(a)-anthracene plus 12-O-tetradecanoylphorbol-13-acetate (TPA)] skin carcinogenesis model. Female transgenic mice expressing the human MnSOD gene in the skin and their nontransgenic counterparts were used in this study. Pathological examination demonstrated significant reduction of papilloma formation in transgenic mice. Quantitative analysis of 4-hydroxy-2-nonenal-modified proteins showed greater accumulation of oxidative damage products in nontransgenic compared with transgenic mice, and this oxidative damage was demonstrated to be present in both mitochondria and nucleus. TPA increased activator protein-1 (AP-1) binding activity within 6 h in nontransgenic mice, but increased AP-1 binding activity was delayed in the transgenic mice. Electrophoretic mobility shift assay, transcription of the target genes, and Western analysis studies indicated that the increased AP-1 binding activity was attributable to induction of the Jun but not the Fos protein families. Overexpression of MnSOD selectively inhibited the TPA-induced activation of protein kinase Cepsilon and prevented subsequent activation of c-Jun NH(2)-terminal kinase in response to TPA. Overall, these results indicate that MnSOD regulates both cellular redox status and selectively modulates PKCepsilon signaling, thereby delaying AP-1 activation and inhibiting tumor promotion, resulting in reduction of tumors in MnSOD transgenic mice.

Overexpression of MnSOD protects murine fibrosarcoma cells (FSa-II) from apoptosis and promotes a differentiation program upon treatment with 5-azacytidine: involvement of MAPK and NFkappaB pathways.

Stable transfection of neomycin and human manganese superoxide dismutase (MnSOD2) expression plasmids into a murine fibrosarcoma cell line (FSa-II) was previously done in our laboratory. Treatment with 10 microM 5-azacytidine induced apoptosis in the control cell line (NEO), whereas the MnSOD-overexpressing cell line (SOD-H) demonstrated differentiated-appearing morphology. The levels of the myogenic transcription factor, MyoD, and the muscle-specific marker, alpha-actin, were increased over time with 5-azacytidine treatment in the SOD-H cell line. Nuclear transcription factor NFkappaB was activated in the SOD-H cell line, whereas inhibition of NFkappaB activation reduced the levels of MyoD and alpha-actin. Members of mitogen-activated protein kinase pathway and the Raf1/MEK/ERK cascade were shown to play a positive role in this event. Overexpression of MnSOD not only can protect cells from the toxic effects of 5-azacytidine, but can also promote the fibrosarcoma cells to enter a differentiation program.

Nuclear factor kappaB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2.

Manganese superoxide dismutase (MnSOD) serves a protective role under conditions of oxidative stress mediated by such diverse agents as adriamycin, radiation, chemical hypoxia and ischaemia and might act as a newly recognized type of tumour-suppressor. MnSOD is an inducible enzyme; however, the signalling molecules and pathways involved in its induction have not been fully elucidated. Recently we reported the identification of a 342 bp enhancer within the second intron (I2E) of the human gene encoding MnSOD (SOD2), which contains sites for binding nuclear factor kappaB (NF-kappaB), CCAAT-enhancer-binding protein (C/EBP) and nuclear factor 1 (NF-1). Using a human fibroblast cell line transformed by simian virus 40, we have identified the I2E fragment as being responsive to PMA. Furthermore, simultaneous treatment with PMA and cytokines (tumour necrosis factor alpha and interleukin 1beta) synergistically increases MnSOD induction. The use of mutant constructs identified the NF-kappaB element within the enhancer fragment as being essential for the PMA and PMA/cytokine effect. Mutations in the C/EBP- and NF-1-binding sites revealed a potential co-operation between proteins that bind to these sites and the NF-kappaB element. Evaluation of inhibitory kappaB (IkappaB)-alpha and IkappaB-beta proteins reveals agent-specific differences in their turnover kinetics. Both C/EBP and NF-kappaB DNA-binding activities were increased in cells receiving a combination of cytokine and PMA. Supershift and immunoprecipitation studies suggest a physical interaction between C/EBP and NF-kappaB proteins. Taken together, these studies suggest the activation of multiple transcription factors as well as pathways leading to increased NF-kappaB activity as being the mechanisms responsible for the synergistic induction of MnSOD by PMA and cytokines.

An intronic NF-kappaB element is essential for induction of the human manganese superoxide dismutase gene by tumor necrosis factor-alpha and interleukin-1beta.

Tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) are cytokines that induce expression of various genes through activation of the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB). We have previously cloned the entire human MnSOD (SOD2) gene and found several NF-kappaB-binding sites in the 5' and 3' flanking and intronic regions. To test whether these putative NF-kappaB-binding sites are able to respond to TNF and IL-1, we performed induction analysis using various deletion constructs ligated to a luciferase reporter gene. We found that the 5' and 3' flanking regions containing several NF-kappaB-binding sites do not mediate MnSOD induction by TNF or IL-1. When a 342-bp intron 2 fragment containing NF-kappaB, C/EBP, and NF-1 binding sites was linked to the basal promoter of the SOD2 gene, transcriptional activities were significantly increased in response to TNF and IL-1 in an orientation- and position-independent manner. To accurately identify the element that is most critical for the enhancer activity, deletions and specific mutations of each individual site were studied. The results indicated that the NF-kappaB binding site is essential but not sufficient for TNF- or IL-1-mediated induction. Furthermore, NF-kappaB elements in the 5' and 3' flanking regions could be made to function in TNF or IL-1 induction when they were transposed to the intronic fragment. Taken together, these results suggest that an NF-kappaB element and its location in the SOD2 gene is critical for TNF/IL-1-mediated induction. However, a complex interaction between NF-kappaB and other transcription elements is needed for a high-level induction.

Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death.

Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesize that mitochondria-derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated murine fibrosarcoma cell lines, which expressed a range of mitochondrial manganese superoxide dismutase (MnSOD) activities, with respiratory chain inhibitors. Apoptosis was confirmed by DNA fragmentation analysis and electron microscopy. MnSOD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl-x(L), p53 and poly(ADP-ribose) polymerase (PARP) to identify specific cellular pathways that might contribute to the mitochondrial-initiated ROS-mediated cell death. Cells overexpressing MnSOD contained less bcl-x(L) within the mitochondria compared to control (NEO) cells, therefore excluding the role of bcl-x(L). p53 was undetectable by Western analysis and examination of the proapoptotic protein bax, a p53 target gene, did not increase with treatment. Activation of caspase-3 (CPP-32) occurred in the NEO cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP-32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fragments. Taken together, these findings suggest that mitochondrial-mediated ROS generation is a key event by which inhibition of respiration causes cell death, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death.

Symposium overview: Characterization of xenobiotic metabolizing enzyme function using heterologous expression systems.

Genetically modified cell lines can be very useful models for assessing the toxicologic effects of modulation of expression of individual gene products in comparison to their isogenic parental control cell lines. This symposium begins with an overview of general issues related to development and utilization of model systems created by transfection of cell lines to induce elevated expression of metabolic enzymes of toxicologic relevance. Selected studies that illustrate the heterologous expression rationale and various approaches to transgenic-cell model construction are represented. Results to date with cells engineered to express specific transfected genes are discussed, with emphasis on the effects of expression of selected phase I or phase II enzymes on cellular sensitivity to several toxic end-points. The individual sections highlight the utility of these model cell lines for examining the role of enzyme catalysis and function in metabolism of biologically active xenobiotic or endobiotic compounds of interest in toxicology. Both activating and detoxifying enzymes are discussed, with principal emphasis on the latter. This symposium includes talks on transfected cells that express aldehyde dehydrogenases, superoxide dismutase, UDP-glycosyltransferases, glutathione transferases, and cytochrome P450 isozymes. In addition to the general toxicologic utility and advantages of these genetically engineered cell lines, this overview emphasizes their particular contributions to the insights obtained to date with the specific model cell lines.

Overexpression of manganese superoxide dismutase selectively modulates the activity of Jun-associated transcription factors in fibrosarcoma cells.

Manganese superoxide dismutase (MnSOD) is reduced in a variety of tumor cells and has been proposed to be a new type of tumor suppressor gene. The mechanism(s) by which MnSOD suppresses cancer development is currently unknown. However, expression of this antioxidant might play a significant role in maintaining cellular redox status. The relationship between MnSOD expression and modulation of DNA-binding activity and transcriptional activation of redox-sensitive oncoproteins and tumor suppressor proteins was studied in a murine fibrosarcoma cell line (FSa-II). Electrophoretic mobility shift assay and transcriptional activation studies revealed an inverse correlation between MnSOD expression and activity of c-jun-associated transcription factors, activator protein 1 and cyclic AMP-responsive element binding protein. Furthermore, expression of an activator protein 1 target gene, bcl-xL, was decreased in MnSOD-transfected cell lines. The results suggest that overexpression of MnSOD may exert its tumor suppressor activity, in part, by modulation of specific oncogenes.